Cell penetrating peptide, conjugate comprising same, and composition comprising conjugate

ABSTRACT

The present invention relates to a conjugate of cell penetrating peptide and an active ingredient; and its use. Specifically, a conjugate including a cell penetrating peptide which is a peptide comprising any one amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156, a fragment of any one sequence of SEQ ID NO: 1 to SEQ ID NO: 156, or a peptide having above 80% homology with the above-mentioned sequence; and a composition comprising the same are disclosed.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name24730810005_SUB_SEQ_LIST.txt; Size: 49,743 bytes; and Date of Creation:Jul. 8, 2015) filed with the application is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to cell penetrating peptides derived fromhuman telomerase reverse transcriptase (hTERT) enzyme, conjugates of thecell penetrating peptides, active ingredients, and compositionscomprising the conjugate.

BACKGROUND

Although Low-molecular weight substances, nucleic acids, proteins,nano-particles, etc, have great potentials as therapeutic substances ata molecular level, their uses are limited due to the incompetence topenetrate into tissues and cell membrane. The development of a system todeliver such substances into the cell has been the active area ofresearch over the last two decades. transport the substances inside thecell has been a conversation topic in a treatment of molecular method.Low-molecular weight substances, nucleic acids or nano-particles weretransported inside the cell by several reagents, electroporation orheatshock. However, it was difficult to find an adequate method ofdelivery of proteins inside the cell without disrupting the activity andintegrity of proteins. In 1980s, in the research conducted on studyingthe cell penetrating capacity of HIV, it was found that HIV-TAT proteinconsisting of specific 11 amino acids play an important role in aprocess of transportation inside the cell. Thus, in 1990s, studies onfinding the right method of transporting proteins inside the cell hasbeen the intense area of research.

Telomere is known as a repetitive sequence of genetic material found atthe ends of chromosomes that prevent chromosomes from damage or mergingonto other chromosomes. The length of the telomere is shortened at eachcell division, and after a certain number of cell division, the telomerelength is extremely shortened to the extent in which the cell stopsdividing and dies. On the other hand, the elongation of telomeres isknown to extend the life span of a cell. For an example, cancer cellsexcrete an enzyme called telomerase, which prevents shortening oftelomeres, thus resulting in proliferation of cancer cells.

The objective of this invention is to provide a novel peptide.

Another objective of present invention is to provide the polynucleotidethat codes the novel peptide.

Another objective of present invention is to provide a cell penetratingpeptide.

Another objective of present invention is to provide a useful peptide asa carrier of the active ingredient in a cell.

Another objective of present invention is to provide a conjugate that anactive ingredient and cell penetrating peptide are conjugated.

Another objective of present invention is to provide a compositioncomprising a conjugate of an active ingredient and cell penetratingpeptide.

Another objective of present invention is to provide a pharmaceuticalcomposition comprising a conjugate of an active ingredient and cellpenetrating peptide.

Another objective of present invention is to provide a functionalcosmetic composition comprising a conjugate of an active ingredient andcell penetrating peptide.

Another objective of present invention is to provide a health foodcomposition comprising a conjugate of an active ingredient and cellpenetrating peptide.

Another objective of present invention is to provide a contrast agentcomprising a conjugate of an active ingredient and cell penetratingpeptide.

SUMMARY OF THE INVENTION

The conjugate according to the one embodiment of the present inventionmay be a conjugate of cell penetrating carrier peptide and activeingredients, wherein the carrier peptide is the peptide comprising anyone amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156, the peptidehaving above 80% homology of amino acid sequence with above-mentionedsequences, or the fragment of the above-mentioned peptides, and whereinthe peptide having above 80% homology of amino acid sequence withabove-mentioned sequences and the fragment of the same are the peptidesthat maintain cell penetrating ability of any one amino acid sequencesof 1 to SEQ ID NO: 156.

According to another embodiment of the conjugate in the presentinvention, the fragment may be made of 3 or more amino acids.

According to another embodiment of the conjugate in the presentinvention, the carrier peptide may be made of 30 or less amino acids.

According to another embodiment of the conjugate in the presentinvention, the above-mentioned carrier peptide may be the peptide havingany one or more amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 156.

According to another embodiment of the conjugate in the presentinvention, which comprises any one amino acid sequence selected from thegroup consisting of: SEQ ID NO: 9, SEQ ID NO: 37, SEQ ID NO: 43, SEQ IDNO: 48, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 90, SEQ ID NO: 92 andSEQ ID NO: 121.

The contrast agent according to the one embodiment of the presentinvention may comprise any one conjugate above-mentioned.

The contrast agent according to the one embodiment of the presentinvention may be for contrasting a cell.

According to another embodiment of the contrast agent in the presentinvention, the cell may be a stem cell.

The composition according to one embodiment of the present invention maycomprise any one of conjugates above-mentioned.

According to another embodiment of the composition in the presentinvention, the active ingredient may be for treatment or prevention ofdisease, and the composition may be pharmaceutical composition.

According to another embodiment of the composition in the presentinvention, the active ingredient may be the active ingredient forfunctional cosmetics, and the composition may be cosmetic composition.

According to another embodiment of the composition in the presentinvention, the active ingredient may be the active ingredient forfunctional health food, and the composition may be health foodcomposition.

The cytoplasm targeting delivery system of active ingredient accordingto the one embodiment of the present invention may comprise any one ofconjugates mentioned above, wherein the carrier peptide moves into acytoplasm locally and performs a role of local cytoplasm delivering thementioned active ingredients, wherein the peptide having above 80%homology of amino acid sequence with above-mentioned sequence and thefragment of the same are the peptides that maintain cell penetratingability of any one amino acid sequences of SEQ ID NO: 1 to SEQ ID NO:156.

According to another embodiment of the method in the present invention,the method may be for delivering the active ingredient locally intomitochondria inside a cell.

According to another embodiment of the cell penetrating peptide in thepresent invention, the above-mentioned carrier peptide may be thepeptide having any one or more amino acid sequences of SEQ ID NO: 1 toSEQ ID NO: 156.

The polynucleotide according to the present invention may encodeabove-mentioned cell penetrating peptide.

The vector according to the present invention may compriseabove-mentioned polynucleotide.

The transformed cell according to the present invention may compriseabove-mentioned vector.

INDUSTRIAL APPLICABILITY

Active ingredients which are difficult to be transported inside a cellcan be easily transported inside a cell by using the peptide, or theconjugate of the peptide and active ingredients, disclosed in thepresent invention. This means that efficacy of active ingredients can beincreased and therefore the dosage can be lowered. As a result, sideeffects due to a drug administration can be minimized and effectivenessof treatment can be increased. Especially, as delivering drugs locallyinto mitochondria, mitochondria related diseases or disorders can beimproved, and the effectiveness of prophylaxis and treatment of diseasescan be increased. In a case of cosmetics, with a small amount of activeingredients, it can create an outstanding effect. By conjugating apeptide with a contrast substance, it can be used as a contrastsubstance to monitor a process of cell transplantation or transplantedcells in a cell treatment. Especially, it can be effectively used as acontrast substance for stem cells injected within a body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 to FIG. 23 represents illustrating that the cellular uptake ofthe cell numbers from a HeLa cell treated with after the FITC fused tothe peptide of SEQ: 1 to SEQ ID NO: 156, and analyzed by FACS. Thecontrol cells were treated only FITC.

FIG. 24 to FIG. 43 represents illustrating that the cellular uptake ofthe cell numbers from a Hurl cell treated with after the FITC fused tothe peptide of SEQ: 1 to SEQ ID NO: 156, and analyzed by FACS. Thecontrol cells were treated only FITC.

FIG. 44 to FIG. 58 represents illustrating that the cellular uptake ofthe cell numbers from a human T lymphocyte cell line (Jurket) treatedwith after the FITC fused to the peptide of SEQ: 1 to SEQ ID NO: 156,and analyzed by FACS. The control cells were treated only FITC.

FIG. 59 to FIG. 72 represents illustrating that the result of toxicityand cell viability from a HeLa cell treated with after the FITC fused tothe peptide of SEQ: 1 to SEQ ID NO: 156, and analyzed by flow cytometry(Flow cytometry). The control cells were treated only FITC.

DETAILED DESCRIPTION OF THE INVENTION

Proteins, Nucleic acids, Peptides or virus etc. have big potentials tobe used as therapeutic substances. However, their uses are limitedbecause they cannot penetrate tissues and cell membrane due to molecularlevel sizes. Although, the size of molecules is small, they cannotpenetrate lipid-bilayer due to structure or characteristics of themolecules. Thus, through the use of electroporation, heat shock, etc.,there were attempts to transport proteins, nucleic acids, peptides orviruses inside the cell; it was difficult to transfer those withoutneither damaging cell membrane nor keeping the active states of abovemolecules. There have been many studies conducted since TAT(Trans-Activating Transcriptional activator) protein derived from HIV(Human Immuno-deficiency Virus) has shown to work as cell penetratingpeptide which can transport huge active substances inside the cell.Specifically, there have been studies conducted about substances thatcan transport huge molecules such as proteins, nucleic acids, peptidesor virus inside the cell without causing any toxicity, unlike TATprotein which causes toxicity inside the cell. Therefore, the presentinvention was completed as present inventors have found that peptidesderived from telomerase have outstanding efficacy as cell penetratingpeptide without a noticeable toxicity.

Peptides are disclosed in SEQ ID NO: 1 to SEQ ID NO: 156 shown in Table1 below. SEQ ID NO: 157 is a full length sequence of the Humantelomerase protein. The “name” in Table 1 below was used for distinctionof peptides. In a different specific embodiment of the presentinvention, more than one peptide of the mentioned peptides in SEQ ID NO:1 to SEQ ID NO: 156 comprise a “synthetic peptide”, a synthesizedpeptide of selected areas of the telomerase. In the presentspecification, the term “pep” herein relates to a peptide that has anyone amino sequence among SEQ ID NO: 1 to SEQ ID NO: 156, or a peptidecomprising of amino acid sequence above 80% homology withabove-mentioned sequences, or a peptide fragment of above-mentionedpeptides.

TABLE 1 SEQ ID Position in NO NAME Telomerase Sequence Length   1. pep2[660-689] ALFSVLNYERARRPGLLGAS 30 aa VLGLDDIHRA   2. pep3 [663-677]SVLNYERARRPGLLG 15 aa   3. pep4 [674-683] PGLLGASVLGL 10 aa   4. pep5[615-624] ALLTSRLRFI 10 aa   5. pep6 [613-621] RPALLTSRL  9 aa   6. pep7[653-661] RLTSRVKAL  9 aa   7. pep8 [691-705] RTFVLRVRAQDPPPE 15 aa   8.pep9 [653-667] RLTSRVKALFSVLNY 15 aa   9. pep10 [651-665]AERLTSRVKALFSVL 15 aa  10. pep11 [667-675] YERARRPGL  9 aa  11. pep12[675-683] LLGASVLGL  9 aa  12. pep13 [680-689] VLGLDDIHRA 10 aa  13.pep14 [677-686] GASVLGLDDI 10 aa  14. pep15 [660-669] ALFSVLNYER 10 aa 15. pep16 [663-672] SVLNYERARR 10 aa  16. pep17 [679-688] SVLGLDDIHR10 aa  17. pep18 [662-671] FSVLNYERAR 10 aa  18. pep19 [665-675]NYERARRPGL 10 aa  19. pep20 [667-676] YERARRPGLL 10 aa  20. pep21[672-681] RPGLLGASVL 10 aa  21. pep22 [668-676] ERARRPGLL  9 aa  22.pep23 [680-688] VLGLDDIHR  9 aa  23. pep24 [663-671] SVLNYERAR  9 aa 24. pep25 [664-672] VLNYERARR  9 aa  25. pep26 [670-678] ARRPGLLGA 9 aa  26. pep27 [673-681] PGLLGASVL  9 aa  27. pep28 [671-679]RRPGLLGAS  9 aa  28. pep29 [660-668] ALFSVLNYE  9 aa  29. pep30[674-682] GLLGASVLG  9 aa  30. pep31 [679-687] SVLGLDDIH  9 aa  31.pep32 [668-675] ERARRPGL  8 aa  32. pep33 [670-677] ARRPGLLG  8 aa  33.pep34 [674-681] GLLGASVL  8 aa  34. pep35 [669-676] RARRPGLL  8 aa  35.pep36 [676-683] LGASVLGL  8 aa  36. pep37 [563-577] VTETTFQKNRLFFYR15 aa  37. pep38 [573-587] LFFYRKSVWSKLQSI 15 aa  38. pep39 [583-597]KLQSIGIRQRLKRYQ 15 aa  39. pep40 [603-617] EAEVRQHREARPALL 15 aa  40.pep41 [613-627] RPALLTSRLRFIPKP 15 aa  41. pep42 [623-637]FIPKPDGLRPIVNMD 15 aa  42. pep43 [643-657] RTFRREKRAERLTSR 15 aa  43.pep45 [683-697] LDDIHRAWRTFVLRV 15 aa  44. pep46 [693-707]FVLRVRAQDPPPELY 15 aa  45. pep47 [721-735] PQDRLTEVIASIIKP 15 aa  46.pep48 [578-592] KSVWSKLQSIGIRQH 15 aa  47. pep49 [593-608]LKVQLRELSEAEVRQ 16 aa  48. pep50  [1-20] MPRAPRCRAVRSLLRSHYRE 20 aa  49.pep51 [21-40] VLPLATEVRRLGPQGWRLVQ 20 aa  50. pep52 [41-60]RGDPAAFRALVAQCLVCYPW 20 aa  51. pep53 [61-80] DARPPPAAPSFRQVSCLKEL 20 aa 52. pep54  [81-100] VARVIARLCERGAKNV1AFG 20 aa  53. pep55 [101-120]FALLDGARGGPPEAFTTSVR 20 aa  54. pep56 [121-140] SYLPNTVTDALRGSGAWGLL20 aa  55. pep57 [141-160] LRRVGDDVLVHLLARCALEV 20 aa  56. pep58[161-180] LVAPSCAYQVCGPPLYQLGA 20 aa  57. pep59 [181-200]ATQARPPPHASGPRRRLGCE 20 aa  58. pep60 [201-220] RAWNHSVREAGVPLGLPAPG20 aa  59. pep61 [221-240] ARRRGGSASRSLPLPKRPRR 20 aa  60. pep62[241-260] GAAPEPERTPVGQGSWAHPG 20 aa  61. pep63 [261-280]RTRGPSDRGFCVVSPARPAE 20 aa  62. pep64 [281-300] EATSLEGALSGTRHSHPSVG20 aa  63. pep65 [301-320] RQHHAGPPSTSRPPRPWDTP 20 aa  64. pep66[321-340] CPPVYAETKHFLYSSGDKEQ 20 aa  65. pep67 [341-360]LRPSELLSSLRPSLTGARRL 20 aa  66. pep68 [361-380] VETIFLGSRPWMPGTPRRLP20 aa  67. pep69 [381-400] RLPQRYWQMRPLFLELLGNH 20 aa  68. pep70[401-420] AQCPYGVLLKTHCPLRAAVT 20 aa  69. pep71 [421-440]PAAGVCAREKPQGSVAAPEE 20 aa  70. pep72 [441-460] EDTDPRRLVQLLRQHSSPWQ20 aa  71. pep74 [481-500] RHNERRFLRNTKKFISLGKE 20 aa  72. pep75[501-520] AKLSLQELIWKMSYRDCAWL 20 aa  73. pep76 [521-540]RRSPGVGCVPAAEHRLREEI 20 aa  74. pep77 [541-560] LAKFLHWLMSVYVVELLRSF20 aa  75. pep78 [561-580] FYVTETTFQKNRLFEYRKSV 20 aa  76. pep79[581-600] WSKLQSIGIRQHLKRVQLRE 20 aa  77. pep80 [601-620]LSEAEVRQHREARPALLTSR 20 aa  78. pep81 [621-640] LREIPKPDGLRPIVNMDYVV20 aa  79. pep82 [641-660] GARTFRREKRAERLTSRVKA 20 aa  80. pep83[661-680] LFSVLNYERARRPGLLGASV 20 as  81. pep85 [701-720]DPPPELYFVKVDVTGAYDTI 20 aa  82. pep86 [721-740] PQDRLTEVIASIIKPQNTYC20 aa  83. pep87 [741-760] VRRYAVVQKAAHGHVRRAFK 20 aa  84. pep88[761-780] SHVSTLTDLQPYMRQFVAHL 20 aa  85. pep89 [781-800]QETSPLRDAVVIEQSSSLNE 20 aa  86. pep90 [801-820] ASSGLEDVFLRFMCHHAVRI20 aa  87. pep91 [821-840] RGKSYVQCQGIPQGSILSTL 20 aa  88. pep92[841-860] LCSLCYGDMENKLFAGIRRD 20 aa  89. pep93 [861-880]GLLLRLVDDFLLVTPHLTHA 20 aa  90. pep94 [881-900] KTFLRTLVRGYPEYGCVVNL20 aa  91. pep95 [901-920] RKTVVNFPVEDEALGGTAFV 20 aa  92. pep96[921-940] QMPAHGLFPITGLLLDTRTL 20 aa  93. pep97 [941-960]EVQSDYSSYARTSIRASLTF 20 aa  94. pep99  [981-1000] KCHSLFLDLQVNSLQTVCTR20 aa  95. pep100 [1001-1020] IYKILLLQAYRFHACVLQLP 20 aa  96. pep101[1021-1040] FHQQVWKNPTFFLRVISDTA 20 aa  97. pep102 [1041-1060]SLCYSILKAKNAGMSLGAKG 20 aa  98. pep103 [1061-1080] AAGPLPSEAVQATCHQAFLL20 aa  99. pep104 [1081-1100] KLTRHRYTYVPLLGSLRTAQ 20 aa 100. pep105[1101-1120] TQLSRKLPGTTLTALEAAAN 20 aa 101. pep106 [1121-1132]PALPSDFKTILD 12 aa 102. pep107  [1-10] MPRAPRCRAV 10 aa 103. pep108[11-30] RSLLRSHYREVLPLATFVRR 20 aa 104. pep109 [31-50]LGPQGWRLVQRGDPAAFRAL 20 aa 105. pep110 [51-70] VAQCLVCVPWDARPPPAAPS20 aa 106. pep111 [71-90] FRQVSCLKELVARVLQRLCE 20 aa 107. pepll2 [91-110] RGAKNYLARGFALIDGARGG 20 aa 108. pep113 [111-130]PPEAFTTSVRSYLPNTVTDA 20 aa 109. pep114 [131-150] LRGSGAWGLLLRRVGDDVLV20 aa 110. pep115 [151-170] HLLARCALFVLVAPSCAYQV 20 aa 111. pep116[171-190] CGPPLYQLGAATQARFPPHA 20 aa 112. pe0117 [191-210]SGPRRRLGCERAWNHSVREA 20 aa 113. pep118 [211-230] GVPLGLPAPGARRRGGSASR20 aa 114. pep119 [231-250] SLPLPKRPRRGAAPEPERTP 20 aa 115. pep120[251-270] VGQGSWAPPGRTRGPSDRGF 20 aa 116. pep121 [271-290]CVVSPARPAEEATSLEGALS 20 aa 117. pep122 [291-310] GTRHSHPSVGRQHPAGPPST20 aa 118. pep123 [311-330] SRPPRPWDTPCPPVYAETKH 20 aa 119. pep124[331-350] FLYSSGDKEQLRPSFLLSSL 20 aa 120. pep125 [351-370]RPSLTGARRLVETIFLGSRP 20 aa 121. pep126 [371-390] WMPGTPRRLPRLPQRYWQMR20 aa 122. pep127 [391-410] PLFLELLGNHAQCPYGVLLK 20 aa 123. pep128 [411-430] THCPLRAAVTPAAGVCAREK 20 aa 124. pepl29 [431-450]PQGSVAAPEEEDTDPRRLVQ 20 aa 125. pep130 [451-470] LLRQHSSPWQVYGEVRACLR20 aa 126. pep131 [471-490] RLVPPGLWGSRPNERRFLRN 20 aa 127: pep132[491-510] TKKFISLGKHAKLSLQELTW 20 aa 128. pep133 [511-530]KMSVRDCAWLRRSPGVGCVP 20 aa 129. pep134 [531-550] AAEHRLREEILAKFLHWLMS20 aa 130. pep135 [551-570] VYVVELLRSFEYVTETTFQR 20 aa 131. pep136[571-590] NRLFFYRKSVWSKLQSIGIR 20 aa 132. pep137 [591-610]QRLKRVQLRELSEAEVRQPR 20 aa 133. pep138 [611-630] EARPALLTSRLRFIPKPDGL20 aa 134. pep139 [631-650] RPIVNMDYVVGARTPRREKR 20 aa 135. pep140[651-670] AERLTSRVKALFSVLNYERA 20 aa 136. pep141 [671-690]RRPGLLGASVLGLDDIHRAW 20 aa 137. pep142 [691-710] RTFVLRVRAQDPPPELYPVK20 aa 138. pep143 [711-730] VDVTGAYDTIPQDRLTEVLA 20 aa 139. pep145[751-770] AHGhVRKAFKSHVSTLTDLQ 20 aa 140. pep146 [771-790]PYMRQFVAHLQETSPLRDAV 20 aa 141. pep147 [791-810] VIEQSSSLNEASSGLFDVEL20 aa 142. pep148 [811-830] RFMCHHAVRIRGKSYVQCQG 20 aa 143. pep149[831-850] IPQGSILSTLLCSLCYGDME 20 aa 144. pep150 [851-870]NKLFAGIRRDGLLLRLVDDF 20 aa 145. pcp151 [871-890] LLVTPHLTHAKTFLRTLVRG20 aa 146. pep152 [891-910] VPLYGCVVNLRKTVVNFEVE 20 aa 147. pep153[911-930] DEALGGTAFVQMPAHGLFEW 20 aa 148. pep154 [931-950]CGLLLDTRTLEVQSDYSSYA 20 aa 149. pep156 [971-990] RRKLFGVLRLKCHSLFLDLQ20 aa 150. pep157  [991-1010] VNSLQTVCTNIYKILLLQAY 20 aa 151. pep158[1011-1030] RFHACVLQLPEHQQVWKNET 20 as 152. pep159 [1031-1050]FFLRVISDTASLCYSILKAK 20 aa 153. pep160 [1051-1070] NAGMSLGAKGAAGPLPSEAV20 aa 154. pep161 [1071-1090] QWLCHQAPILKLTRHRVTYV 20 aa 155. pep162[1091-1110] PLLGSLRTAQTQLSRKLEGT 20 aa 156. pep163 [1111-1132]TLTALEAAANPALPSDFKTI 22 aa LD 157. Telo-    [1-1132]MPRAPRCRAVRSLLRSEREY 1132 merse LPLATHRRLGEQGWRLVQRGDPAAFRALVAQCLVCVPWDA RPPPAAPSFRQVSGLKELVA RVLQRLCERGAKNVLAFGEALLDGARGGPPEAFTTSVRSY LENIVTDALRGSGAWGLLLR RVGDDVLWILLARCALFVLVAPSCAYQVCGPPLYQLGAAT QARPEEHASGRRRLGCERAW NHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAA PEPERTPVGQGSWAHPGRTR GRSDRGFCVVSFARPAERATSLECALSGTRHSHPSVGRWH AGPPSTSRPERPIVDTECPP VYAETKHFLYSSGDREQLRPSELLSSLRPSLTGARRLVET IFLGSRPMEGTPRRLPRLPQ RYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAC VCAREKPQGSVAAPEEEDTD PRITLVQLLRQMSSPWQVYGEVRACLRRLVITGLIMSENE RRELRNTKKFISLGKLIAKL SLQELTWKMSVRDCAWLRRSEGVGCVPAARHRLREEILAK FLHWLMSVYVVELLRSFEYV TETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSE AFVRQHREARPALLTSRLRF IPKPDGLRPIVNMDYVVGARTFERERRAERLTSRVRALFS VLNYERARRPGLLGASVLGL DDIHRAWRTFVLRVRAQDEPPELVFVKVDVTGAYDTIPUR LTEVIASIIKPQNTYCVRRY AVVQKAAHGHVRKAFKSHVSTLTDLQEVMRQFVAHLQFTS PLRDAVVJEQSSSLNEASSG LFDVELRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSL CYGDMENKLFAGIRRDGLLL RLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTV VNFPVEDEALGGTAFVQMPA HGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGF EAGRNMRRKLEGVLRLKCHS LELDLQVNSLQTVCTNIYKILLWAYRFHACVLQLPFHQQV WKNPTFFLRVISDTASLCYS ILKAKNAGMSLGAKGAAULPSEAVQWLCMQAFLLKLTRHR VTYVPLLGSLRTAQTQLSRK LPGTTLTALEAAANPALPSD EKTILD164  pep1 [611-626] EARPALLTSRLRFIEK 16 aa

In one embodiment of the present invention the polynucleotide codes apeptide comprising at least one amino acid sequence of SEQ ID NO: 1 toSEQ ID NO: 156, a peptide having above 80% homology with above-mentionedsequences, or a peptide being a fragment of the above-mentionedpeptides. The polynucleotide mentioned above enables production of thepeptides in large quantities. For example, cultivation of vectors thatinclude polynucleotides encoding peptides allows production of peptidesin large quantities.

The peptides disclosed herein can include a peptide comprising aminoacid sequence above 80%, above 85%, above 90%, above 95%, above 96%,above 97%, above 98%, or above 99% homology. Moreover, the peptidesdisclosed in the present invention can include a peptide comprising anyone amino sequence among SEQ ID NO: 1 to SEQ ID NO: 156 or itsfragments, and a peptide with more than 1 transformed amino acid, morethan 2 transformed amino acid, more than 3 transformed amino acid, morethan 4 transformed amino acid, more than 5 transformed amino acid, morethan 6 transformed amino acid, or more than 7 transformed amino acid.

In one embodiment of the present invention, changes in amino acidsequence belong to the modification of peptide's physical and chemicalcharacteristics. For example, amino acid transformation can be performedby improving thermal stability of the peptide, altering substratespecificity, and changing the optimal pH.

The term “amino acid” herein includes not only the 22 standard aminoacids that are naturally integrated into peptide but also the D-isomersand transformed amino acids. Therefore, in a specific embodiment of thepresent invention, a peptide herein includes a peptide having D-aminoacids. On the other hand, a peptide may include non-standard amino acidssuch as those that have been post-translationally modified. Examples ofpost-translational modification include phosphorylation, glycosylation,acylation (including acetylation, myristoylation, palmitoylation),alkylation, carboxylation, hydroxylation, glycation, biotinylation,ubiquitinylation, transformation in chemical properties (e.g. β-removingdeimidation, deamidation) and structural transformation (e.g. formationof disulfide bridge). Also, changes of amino acids are included and thechanges of amino acids occur due to chemical reaction during thecombination process with crosslinkers for formation of a peptideconjugate.

A peptide disclosed herein may be a wild-type peptide that has beenidentified and isolated from natural sources. On the other hand, whencompared to peptide fragments of any one amino sequence among SEQ ID NO:1 to SEQ ID NO: 156, the peptides disclosed herein may be artificialmutants that comprise one or more substituted, deleted and/or insertedamino acids. Amino acid alteration in wild-type polypeptide—not only inartificial mutants—comprises conservative substitution of amino acidsthat do not influence protein folding and or activation. Examples ofconservative substitution belong to the group consisting of basic aminoacids (arginine, lysine and histidine), acidic amino acids (glutamicacid and aspartic acid), polar amino acids (glutamine and asparagines),hydrophobic amino acids (leucine, isoleucine, valine and methionine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, and threonine). The amino acidsubstitutions that do not generally alter the specific activity areknown in the art of the present invention. Most common occurredalteration are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, Asp/Gly, and the opposite alterations. Another exampleof conservative substitutions are shown in the following table 2.

TABLE 2 Original amino Preferable residue acid Examples of residuesubstitution substitution Ala (A) val; leu; ile Val Arg (R) lys; gln;asn Lys Asn (N) gln; his; asp, lys; arg Gln Asp (D) glu; asn Glu Cys (C)ser; ala Ser Gln (Q) asn; glu Asn Glu (E) asp; gln Asp Gly (G) ala AlaHis (H) asn; gln; lys; arg Arg Ile (I) leu; val; met; ala; phe;norleucine Leu Leu (L) norleucine; ile ; val; met; ala; phe Ile Lys (K)arg; gln; asn Arg Met (M) leu; phe; ile Leu Phe (F) leu; val; ile; ala;tyr Tyr Pro (P) ala Ala Ser (S) thr Thr Thr (T) ser Ser Trp (W) tyr; pheTyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; ala;norleucine Leu

The substantial transformation of the biological properties of peptidesare performed by selecting significantly different substitution in thefollowing efficacies: (a) the efficacy in maintaining the structure ofthe polypeptide backbone in the area of substitution, such as sheet orhelical three-dimensional structures, (b) the efficacy in maintainingelectrical charge or hydrophobicity of the molecule in the target area,or (c) the efficacy of maintaining the bulk of the side chain. Naturalresidues are divided into groups by general side chain properties as thefollowing:

-   (1) hydrophobicity: Norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilicity: cys, ser, thr;-   (3) acidity: asp, glu;-   (4) basicity: asn, gln, his, lys arg;-   (5) residue that affects chain orientation: gly, pro; and-   (6) aromaticity: trp, tyr, phe.

Non-conservative substitutions may be performed by exchanging a memberof the above classes to different classes. Any cysteine residues thatare not related in maintaining the proper three-dimensional structure ofthe peptide can typically be substituted into serine, thus increasingthe oxidative stability of the molecule and preventing impropercrosslinkage. Conversely, improvement of stability can be achieved byadding cysteine bond(s) to the peptide.

Altered types of amino acids variants of peptides are those thatantibody glycosylation pattern changed. The term “change” herein relatesto deletion of at least one carbohydrate residues that are found in apeptide and/or addition of at least one glycosylated residues that donot exist within a peptide

Glycosylation in peptides are typically N-connected or O-connected. Theterm “N-connected” herein relates to that carbohydrate residues areattached to the side chain of asparagine residues. As tripeptidesequences, asparagine-X-serine and asparagine-X-threonine (where the Xis any amino acid except proline) are the recognition sequence forattaching carbohydrate residue enzymatically to the side chain ofasparagine. Therefore, with the presence of one of these tripeptidesequences in a polypeptide, the potential glycosylation sites arecreated. “O-connected glycosylation” means attaching one of sugarN-acetylgalactosamine, galactose, or xylose to hydroxyl amino acids. Thehydroxyl amino acids are most typically serine or threonine, but5-hydroxyproline or 5-hydroxylysine can be used.

Addition of glycosylation site to a peptide is conveniently performed bychanging amino acid sequence to contain tripeptide sequence mentionedabove (for N-linked glycosylation sites). These changes may be made byaddition of at least one serine or threonine residues to the firstantibody sequence, or by substitution with those residues (for O-linkedglycosylation sites).

In one embodiment of the present invention, cell penetrating peptidecomprising any one amino acid sequences of SEQ ID NO: 1 to SEQ ID NO:156, a peptide having above 80% homology of amino acid sequence withabove-mentioned sequences, or a fragment of the above-mentionedpeptides, is provided.

In one embodiment of the present invention, a pharmaceutical compositioncomprising peptide as a drug delivery system to transport more than oneactive ingredient is provided, wherein the peptide comprises any oneamino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156, the peptide hasabove 80% homology with above-mentioned sequence, or the peptide is afragment of above-mentioned peptide.

A peptide comprising any one amino acid sequence of SEQ ID NO: 1 to SEQID NO: 156, a fragment of above-mentioned peptide, or a peptide havingabove 80% homology with above-mentioned sequence, is safe and hasoutstanding efficacy as cell penetrating peptide. Therefore, the peptidecan be conjugated with a drug to transport the drug inside the cell.

In one embodiment of the present invention, a conjugate of a peptide andan active ingredient to be transported is provided, wherein the peptidecomprises any one amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156,the peptide is a fragment of above-mentioned peptide, or the peptide hasabove 80% homology with above-mentioned peptide. In one embodiment ofthe present invention, an active ingredient may be at least one selectedfrom proteins, nucleic acids, peptides, lipids, glycolipids, minerals,sugars, contrast substances, drugs and chemical compounds. In oneembodiment of the present invention, the active ingredients may bepeptides. In one embodiment of the present invention, the activeingredients may be cytokines, antibody, antibody fragments, therapeuticenzymes, soluble receptors, or ligands.

A cell penetrating peptide disclosed herein means a peptide which cantransport cargo from in vitro and/or in vivo to inside the cell. A“cargo” disclosed herein comprises all the substances that can betransported inside the cell via conjugation with a cell penetratingpeptide, For example, all the substances which want to increase cellpenetrating efficacy, specifically drugs, cosmetics, or activeingredients of health food, more specifically substances which cannot betransported inside the cell via general route, more specifically,sugars, nano-particles, biological formulation, viruses, contrastsubstances or other chemical compounds which can have proteins, nucleicacids, peptide, minerals, glucose as an example, but not limited tothose. A “drug” disclosed herein is a broad concept including asubstance to be transported for alleviation, prophylaxis, treatment ordiagnosis of diseases, wounds, or specific symptom.

A “carrier peptide” disclosed herein is a peptide which can transportactive ingredients to a targeted site via conjugation with activeingredients.

In one embodiment of the present invention, protein or peptide as acargo comprises one or more of hormone, hormone analogue, enzyme, enzymeinhibitors, signal transfer proteins (or peptides), antibody andvaccine, but not limited to those. In one embodiment of the presentinvention, a nucleic acid is a molecule that can be spontaneous orartificial DNA or RNA molecules, either single-stranded ordouble-stranded. The nucleic acid molecule can be one or more nucleicacids of same type (for example, having a same nucleotide sequence) ornucleic acids of different types. The nucleic acid molecules compriseone or more DNA, cDNA, decoy DNA, RNA, siRNA, miRNA shRNA, stRNA,snoRNA, snRNA PNA, antisense oligomer, plasmid and other modifiednucleic acids, but not limited to those. In one embodiment of thepresent invention, virus comprises the whole virus or the core of viruswhich includes nucleic acids of the virus. In one embodiment of thepresent invention, a chemical substance is a broad indication comprisinga natural or synthetic substance which can act as a drug.

In one embodiment of the present invention, drug to be delivered intocells by a cell-permeable peptide is iposomes, micelles, nanoparticles,or quantum dots as drug delivery.

The term “contrast substance” disclosed herein is a broad indicationcomprising all the substances used to contrast structures or fluidswithin the body in medical imaging. An appropriate contrast substancecomprises radiopaque contrast agent, paramagnetic contrast agent,superparamagnetic contrast agent, CT (computed tomography) and othercontrast substances, but not limited to those. For example, a radiopaquecontrast agent (for x-ray imaging) will comprise inorganic iodinecompound and organic iodine compound (for example, diatrizoate),radiopaque metals and their salts (for example, silver, gold, platinum,etc.) and other radiopaque compounds (for example, calcium salts, bariumsalt such as barium sulfate, tantalum and oxidized tantalum). Anappropriate paramagnetic contrast substance (for MR imaging) comprisesgadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and itsderivatives, other gadolinium, manganese, iron, dysprosium, copper,europium, erbium, chrome, nickel and cobalt complex, for example,1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA),ethylenediaminetetraacetic acid (EDTA),1,4,7,10-tetraazacyclododecan-N,—N′,N″-triacetic acid (DO3A),1,4,7-triazacyclononane-N,N′, N″-TRIACETIC ACID (NOTA),1,4,8,10-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),hydroxybenzylethylene-diamine diacetic acid (HBED). An appropriatesuperparamagnetic contrast substance (for MR imaging) comprisesmagnetite, superparamagnetic iron oxide (SPIO), ultrasmallsuperparamagnetic iron oxide (USPIO) and monocrystalline iron oxide.Other appropriate contrast substances are iodinated, non-iodinated,ionic and non-ionic CT contrast agents, a contrast substance likespin-label or diagnostically effective agent.

Other examples of contrast substances comprise β-galactosidase, GreenFluorescent Protein, Cyan Fluorescent Protein, luciferase, but notlimited to those, and a marker gene which codes for protein which can beeasily detected when expressed within cells. Various labels such asradionuclide, flour, enzyme, enzyme-substrate, enzyme cofactor, enzymeinhibitor, ligands (especially hapten) can be used.

In one example of the present invention, a contrast substance isferrocenecarboxylic acid of the below chemical formula 2. The structureof ferrocene is shown in the chemical formula 1.

In one example of the present invention, a conjugate of cell penetratingpeptide and a contrast substance is Ferrocenecarboxylic-pep(Ferrocenecarboxylic-pep) shown in the below chemical formula 3.

In one embodiment of the present invention, a peptide or composition canbe fused with one or more detectable labels. Labels may be compoundswhich can be detected in chemical, physical or enzymatic responses, orcompounds which generate signals directly or indirectly in theresponses. Labeling and detecting after then can be performed accordingto the known method in the art (For example, Sambrook, J., and Russel,D. W. (2001); and Lottspeich, F., and Zorbas H. (1998) Bioanalytik,Spektrum Akademischer Verlag, Heidelberg/Berlin, Germany). Labelscomprise fluorescent label, enzyme label, chromogenic label,luminescence label, radiation label, hapten, biotin, metal complex,metal and colloidal gold, but not limited to those. All forms of theselabels are well known in this field of work, they can be commerciallyobtained from various suppliers.

In one embodiment of the present invention, a cargo can be directlycombined with the peptide. In another embodiment of the presentinvention, a cargo can be combined to the peptide via various types ofbonds such as covalent or non-covalent bonds. A cargo, for example, canbe combined to the N-terminal or C-terminal of the peptide in oneembodiment of the present invention. For example, a cargo can be bondedto the peptide by disulfide bonds or covalent bonds. The covalent bondsare the bonds that a cargo can be bonded to α-amine of N-terminalglutamate, or amine of C-terminal Lysine residues. Also, a peptide and acargo can be combined via a non-covalent bond, which can have either apeptide or a cargo can encapsulate the other as a capsule form.

In another embodiment of the present invention, a peptide can becombined with a cargo via a linker. For example, a peptide can becombined with a cargo by binding a cargo to a linker after introducing alinker such as Hynic(6-hydrazinopyridine-3-carboxylic acid) linker, tothe α-amine of N-terminal glutamate, or amine of C-terminal Lysineresidues.

In another embodiment of the present invention, when a cargo is DNA orRNA, SH group (thiol group) is introduced to the peptide, and maleimidegroup is introduced to DNA or RNA, then, SH group of the peptide andmaleimide group of DNA or RNA are combined, thus creating a bond betweenthe cargo and the peptide.

In another embodiment of the present invention, when a cargo is apeptide or protein, DNA which expresses a cargo is combined with DNAwhich expresses a carrier peptide, and by expressing this, a cargo and apeptide can be combined as a form of fusion protein. Specific examplesof combination by a fusion protein are as follows: when manufacturingprimer for production of fusion protein, a nucleotide coding a carrierpeptide is attached in front of a nucleotide expressing a cargo, and theobtained nucleotide is inserted to a vector such as pET vector using arestriction enzyme, and the nucleotide is expressed by transformationinto a cell such as BL-21(DE3). At this time, a fusion protein is to beeffectively expressed by treating it with an expression inducing agentlike IPTG (isopropyl-1-thio-β-D-galactopyranoside). Then, the expressedfusion protein is purified by His tag purification, and is dialyzed withPBS, and is added to a kit to be concentrated by centrifugation undersuch condition for 5 to 20 mins at 2,000 to 4,000 rpm.

In one embodiment of the present invention, a carrier peptide iscombined with dying substances, fluorescent substances, specificallyFITC (fluorescein isothiocyanate) or GFP (Green Fluorescent Protein). Inone embodiment of the present invention, FITC is combined with aminogroup (NH³⁺) of lysine at N-terminal or C-terminal of a carrier peptide.In the case of a peptide, where lysine does not exist at its terminal,the peptide can be combined with FITC via a linker including Lysine.

The carrier peptide disclosed herein which is the peptide comprising anyone amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156, or thepeptide having above 80% homology of amino acid sequence withabove-mentioned peptides, or a fragment of above-mentioned peptide, canbe combined with a cargo at a mole fraction of 1:1, but it can becombined at mole fraction other than 1:1. For example, a mole fractionof CPP and a cargo may be more than 2:1, specifically, more than 2:1,more than 3:1, more than 4:1, more than 5:1, more than 6:1, more than7:1, more than 8:1, more than 9:1 or more than 10:1. This means thatnumerous carrier peptide molecules can be combined with a cargomolecule. The numerous carrier peptide molecules can be combined inseries or in parallel. “Combined in series” means that a carrier peptideand a cargo molecule are to be combined at terminal amino acids.“Combined in parallel” means that they are to be combined at a siteother than terminal amino acids. On the other hand, the mole fraction ofa carrier peptide and a cargo may be more than 1:2. This means that acarrier peptide molecule can be combined with numerous number of a cargomolecule. For example, a mole fraction of a carrier peptide and a cargomay be 1:2, specifically, more than 1:2, more than 1:3, more than 1:4,more than 1:5, more than 1:6, more than 1:7, more than 1:8, more than1:9 or more than 1:10.

A movement pathway of the peptide combined with Fluoresceinisothiocyanate can be easily found. Therefore, a carrier peptide in oneembodiment of the present invention is to be used for cell imaging ordetecting a pathway of drug delivery inside a cell.

In one embodiment of the present invention, a use of the peptide as adrug delivery carrier to transport more than one active ingredient isprovided, wherein the peptide comprises any one amino acid sequence ofSEQ ID NO: 1 to SEQ ID NO: 156, or the peptide is a fragment ofabove-mentioned peptide, or the peptide has above 80% homology of aminoacid sequence with above-mentioned peptide. The use may indicatetherapeutic or non-therapeutic use.

In one embodiment of the present invention, a method of delivering drugsinside a cell of a subject comprising a step of administering acomposition comprising a drug; and the peptide is provided; wherein thepeptide comprises any one amino acid sequence of SEQ ID NO: 1 to SEQ IDNO: 156, or the peptide is a fragment of above-mentioned peptide, or thepeptide has above 80% homology of amino acid sequence withabove-mentioned peptide.

In one embodiment of the present invention, a method of detecting drugdelivery pathway comprising a step of applying the peptide and acontrast substance to a subject is provided; wherein the peptidecomprises any one amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156,or the peptide is a fragment of above-mentioned peptide, or the peptidehas above 80% homology of amino acid sequence with above-mentionedpeptide.

In one embodiment of the present invention, a method of detecting drugdelivery pathway comprising a step of applying of the conjugate of thepeptide and a contrast substance to a subject is provided; wherein thepeptide comprises any one amino acid sequence of SEQ ID NO: 1 to SEQ IDNO: 156, or the peptide is a fragment of above-mentioned peptide, or thepeptide has above 80% homology of amino acid sequence withabove-mentioned peptide.

In one embodiment of the present invention, a kit for drug delivery intoa cell of a subject containing the composition and an instruction isprovided, wherein the composition comprises a conjugate of a peptide ofthe invention and a drug for delivery, wherein the peptide comprises anyone amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156 or the peptideis a fragment of above-mentioned peptide, or the peptide has above 80%homology of amino acid sequence with above-mentioned peptide, whereinthe instruction includes at least one of administration dose,administration route, administration frequency, and indication of thecomposition.

In one embodiment of the present invention, cosmetic or food compositioncomprising an active ingredient; and the peptide is provided; whereinthe peptide comprises amino acid sequence of SEQ ID NO: 1 to SEQ ID NO:156, the peptide has above 80% homology of amino acid sequence withabove-mentioned sequence, or the peptide is a fragment of theabove-mentioned peptides. In another embodiment of the presentinvention, cosmetic or food composition comprising a conjugate of thepeptide and active ingredients is provided; wherein the peptidecomprises any one amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156,the peptide has above 80% homology of amino acid sequence withabove-mentioned sequence, or the peptide is a fragment of theabove-mentioned peptides.

In one embodiment of the present invention, pharmaceutical, cosmetic orfood composition with an outstanding ability to transport activeingredients inside a cell, comprising a conjugate of the peptide and anactive ingredient, is provided; wherein the peptide comprises any oneamino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 156, the peptide hasabove 80% homology of amino acid sequence with above-mentioned sequence,or the peptide is a fragment of the above-mentioned peptides.

Mitochondria, as a central organelle in energy metabolism of aeukaryotic cell, is a first known intracellular organelle to be relatedto human diseases (Luft R, Ikkos D, Palmieri G, Ernster L, Afzelius B: Acase of severe hypermetabolism of non thyroid origin with a defect inthe maintenance of mitochondrial respiratory control: a correlatedclinical, biochemical, and morphological study, J Clin Invest 41:1776-804, 1962).

Since the mitochondria play an important role in control of energymetabolism of cell and apoptosis, they act as a major target for varioustherapeutic drugs. Also, this organelle is involved in control of thecalcium concentration inside the cell, the mitochondrial respiratorychain acts as an electron transport system which is important in energyproduction, and it causes production of reactive oxygen species. As aresult, the abnormal mitochondrial function has a close relationshipwith adult diseases such as diabetes, cardiomyopathy, infertility,blindness, renal/liver diseases, and stroke (Modica-Napolitano K S.Singh K K: April mitochondria as targets for detection and treatment ofcancer. Expert Rev Mol Med 11:1-19, 2002). Also, it is being suggestedthat Mitochondrial genetic mutations to be involved in the outbreak ofaging, degenerative neuronal disease and cancer etc.

The mitochondria targeting delivery system can be provided according tothe one embodiment of the present invention may comprise any one ofconjugates mentioned above, wherein the carrier peptide moves intointracellular mitochondria locally and performs a role of localintracellular mitochondria delivering the mentioned active ingredients,wherein the peptide having above 80% homology of amino acid sequencewith above-mentioned sequence and the fragment of the same are thepeptides that maintain mitochondria targeting delivery system, theabove-mentioned mitochondria targeting peptide may be the peptide havingany one amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 156.

The mitochondria activity adjusting composition can be provided, whereinthe composition comprises a conjugate of a peptide of the invention anda carried peptide for delivery, wherein the carrier peptide moves intointracellular mitochondria locally and performs a role of localintracellular mitochondria delivering the mentioned active ingredients,wherein the peptide having above 80% homology of amino acid sequencewith above-mentioned sequence and the fragment of the same are thepeptides that maintain mitochondria targeting delivery system, theabove-mentioned mitochondria targeting peptide may be the compositionhaving any one amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 156.

The mitochondria activity adjusting composition according to the oneembodiment of the present invention, wherein the composition for thetreatment of a mitochondrial related diseases or disorder, prevention,inhibitory of progress, or relief of symptoms as an a pharmaceuticalcomposition, wherein the active ingredient will be treated for amitochondrial related diseases or disorder, prevention, inhibitory ofprogress, or relief of symptoms.

The “Mitochondrial related diseases” disclosed herein compriseHuntington's disease, amyotriophic lateral sclerosis, MELAS(Mitochondrial Encephalomyopathy with Lactic Acidemia and Stroke-likeepisodes); MERRF (Myoclonus, epilepsy, and myopathy with ragged redfibers; NARP/MILS (Neurogenic muscular weakness, ataxia, retinitispigmentosa/Maternally inherited leigh syndrome); LHON (Lebers hereditaryoptic neuropathy); KSS (Kearns-Sayre Syndrome); PMPS (PearsonMarrow-Pancreas Syndrome); CPEO (Chronic progressive externalopthalnoplegia); Reye's syndrome; Alper's syndrome; Multiple mtDNAdeletion syndrome; mtDNA depletion syndrome; Complex I deficiency;Complex II (SDH) deficiency; Complex III deficiency; Cytochrome coxidase (COX, Complex IV) deficiency; Complex V deficiency; Adeninenucleotide translocator (ANT) deficiency; Pyruvate dehydrogenase (PDH)deficiency; Ethyl malonic acid aciduria having lactic acid acidemia;3-methyl glutaconic acid aciduria having lactic acid acidemia;refractoriness epilepsy representing a decline during infection;Asperger's syndrome representing a decline during infection; Autismrepresenting a decline during infection; Attention deficit hyperactivitydisorder (ADHD); Cerebral palsy representing a decline during infection;Alexia representing a decline during infection; Maternal hereditarythrombocytopenia; Leukemia; MNGIE (Mitochondrial myopathy, peripheraland autonomic neuropathy, gastrointestinal dysfunction, and epilepsy);MARIAHS syndrome (Mitochondrial ataxia, recrudescent infection, aphasia,hypouricemia/hypomyelination, seizure and dicarboxylic acid aciduria);ND6 dystonia; Cyclic vomiting syndrome representing a decline duringinfection; 3-hydroxyisobutyric acid aciduria having lactic acidacidemia; Diabetes having lactic acid acidemia; Uridine reactive neuralsyndrome (URNS); Familial bilateral striatum necrosis (FBSN); Hearingloss related with aminoglycoside; Relaxed myocardiopathy; Spleenlymphoma; Wolframs syndrome; Multiple mitochondria DNA deletionssyndrome; and Renal tubular acidosis/diabetes/ataxia syndrome, but notlimited to those.

In another embodiment of the present invention, nucleic acid moleculesencoding above-mentioned polypeptides are provided. The nucleic acidmolecules, for example, have base sequences of GAA GCG CGC CCG GCG CTGCTG ACC AGC CGC CTG CGC TTT ATT CCG AAA (SEQUENCE NO: 181). The nucleicacids can be introduced into the host cell according to a known methodto those skilled in the art. For example, the known methods may betransformation method by calcium phosphate method, liposome,electroporation, contacting a virus and a cell, or micro injectiondirectly into the cell, etc. The host cell is higher eukaryotic cell,for example, mammalian cells, or lower eukaryotic cells, such as a yeastcell, or prokaryotic cells, such as a bacterial cell. The prokaryotichost cells appropriate for transformation may be the species whichbelong to E. coli, Bacillus subtillis, Salmonella typhimurium,Pseudomonas, Streptomyces, and Micro bacteria species, as examples.

The vector including above-mentioned nucleic acid molecules is generallyrecombinant expression vector and it comprises, origin of replicationenabling a host cell transformation, and a selectable marker (forexample, dihydrofolate reductase for eukaryotic cell culture, ortolerance of neomycin, tolerance of tetra-cycline or ampicillin in E.coli, or S. cerevisiae TRP1 gene), and the promoter for controllingtranscription of protein coating sequences. Available expression vectorsare, for example, known bacterial plasmids such as SV40, derivatives ofpcDNA, and known bacterial plasmids such as colE1, pCR1, pBR322,pMal-C2, pET, pGEX (Smith, et al., Gene 67:31-40 (1988)), plasmids suchas pMB9 and its derivative RP4, phage DNA which is the same as numerousderivatives of phage I such as NM989, phage DNA such as M13 andsingle-stranded phage DNA of filament type; yeast plasmid, for example,phage DNA or vector induced from a combination of modified plasmid forusing expression suppression sequences and phage DNA. The mammalianexpression vectors comprise origin of replication, an appropriatepromoter and an enhancer. Also, they can comprise compulsory ribosomebinding sites, polyadenylation sites, splice donor, and receptor part,transcription termination sequences, and 5′ planking non-transcriptionalsequences. The mammalian expression vectors can comprise an induciblepromoter, for example, a vector containing dihydrofolate reductasepromoter, any expression vectors containing DHFR expression cassette orDHFR/methotrexate co-amplification vector such as pED. (Randal J,kaufman, 1991, Randal J. Kaufman, Current Protocols in MolycularBiology, 16, 12(1991)). Or, glutamine synthetase/methionine sulfoximineco-amplification vector, for example, pEE14 (Celltech),Epstein-Barr-Virus (EBV), or a vector directing episomal expressionunder the control of nuclear antigen (EBNA), for example, pREP4(Invitrogen), pCEP4 (Invitrogen), pMEP4 (Invitrogen), pREP8(Invitrogen), pREP9 (Invitrogen) and pEBVH is (Invitrogen) can be used.Selectable mammalian expression vectors are Rc/CMV (Invitrogen) andpRc/RSV (Invitrogen) etc. Vaccinia virus mammalian expression vectorswhich can be used in the present invention are pSC11, pMJ601, pTKgptF1S,etc.

Yeast expression vector system to be used in the present invention isnon-fusion pYES2 vector (Invitrogen), fusion pYESHisA, B, C(Invitrogen), pRS vector, etc.

The above-mentioned vectors can be introduced to various cells, such asmammalian cells which is especially the human derived cells, orbacteria, yeast, fungi, insects, nematodes, and plant cells. Theexamples of appropriate cells are VERO cell, HELA cell, for example,ATCC No. CCL2, CHO cell line, for example, ATCC No. CCL61, COS cell, forexample COS-7 cell and ATCC No. CRL 1650 cell, W138, BHK, HepG2, 3T3,for example, ATCC No. CRL6361, A549, PC12, K562 cell, 293 cell, Sf9cell, for example, ATCC No. CRL1711 and Cv1 cell, such as ATCC No.CCL70, etc.

Other appropriate cells to be used in the present invention areprokaryotic host cell strain, for example, the strains belonging to E.coli (e.g. DH5-α strain), Bacillus subtilis, Salmonella typhimurium,Pseudomonas, Streptomyces and Staphylococcus.

In one embodiment of the present invention, the composition may contain0.1 μg/mg to 1 mg/mg, specifically 1 μg/mg to 0.5 mg/mg, morespecifically 10 μg/mg to 0.1 mg/mg of a peptide comprising any one aminoacid sequence of SEQ ID NO: 1 to SEQ ID NO: 156, a peptide comprisingamino acid sequence above 80% homology with above-mentioned sequence, ora fragment of above-mentioned peptide. When the peptide is contained inthe above-mentioned range, all the safety and stability of thecomposition can be satisfied and appropriate in terms ofcost-effectiveness.

In one embodiment of the present invention, the composition may haveapplication with all animals including human, dog, chicken, pig, cow,sheep, guinea pig, and monkey.

In one embodiment of the present invention, the pharmaceuticalcomposition may be administered through oral, rectal, transdermal,intravenous, intramuscular, intraperitoneal, in the bone marrow,epidural or subcutaneous means.

Forms of oral administration may be, but not limited to, tablets, pills,soft or hard capsules, granules, powders, solution, or emulsion. Formsof non-oral administration can be, but not limited to, injections,drips, lotions, ointments, gels, creams, suspensions, emulsions,suppository, patch, or spray.

In one embodiment of the present invention, the pharmaceuticalcomposition, if necessary, may contain additives, such as diluents,excipients, lubricants, binders, disintegrants, buffers, dispersants,surfactants, coloring agents, aromatics or sweeteners.

In one embodiment of the present invention, the pharmaceuticalcomposition may be manufactured by conventional methods of the industryin the art.

In one embodiment of the present invention, the active ingredient of themedical composition may vary according to the patient's age, sex,weight, pathology and state, administration route, or prescriber'sjudgment. Dosage based on these factors is determined within levels ofthose skilled in the art, and the daily dose for example may be, but notlimited to, 0.1 μg/kg/day to 1 g/kg/day, specifically 1 μg/kg/day to 10mg/kg/day, more specifically the 10 μg/kg/day to 1 mg/kg/day, morespecifically the 50 μg/kg/day to 100 μg/kg/day. In one embodiment of thepresent invention, the pharmaceutical composition may be administered,but not limited to, 1 to 3 times a day.

In one embodiment of the present invention, cosmetic composition may beprovided in all forms appropriate for topical applications. For example,forms may be provided as solutions, emulsions obtained by dispersion ofoil phase in water, emulsion obtained by dispersion of water in oilphase, suspension, solid, gel, powder, paste, foam or aerosol. Theseforms may be manufactured by conventional methods of the industry in theart.

In one embodiment of the present invention, the cosmetic composition mayinclude, within levels that won't harm the main effect, otheringredients that may desirably increase the main effect. In oneembodiment of the present invention, the cosmetic composition mayadditionally include moisturizer, emollient agents, surfactants, UVabsorbers, preservatives, fungicides, antioxidants, pH adjusting agent,organic or inorganic pigments, aromatics, cooling agent orantiperspirant. The formulation ratio of the above-mentioned ingredientsmay be decided by those skilled in the art within levels that won't harmthe purpose and the effects of the present invention, and theformulation ratio based on total weight of the cosmetic composition maybe 0.01 to 5% by weight, specifically 0.01 to 3% by weight.

In one embodiment of the present invention, food composition is notlimited to forms, but for example may be granules, powder, liquid, andsolid forms. Each form may be formed with ingredients commonly used inthe industry appropriately chosen by those skilled in the art, inaddition to the active ingredient, and may increase the effect withother ingredients.

Decision for dosage on the above-mentioned active ingredient is withinthe level of those skilled in the art, and daily dosage for example maybe 1 μg/kg/day to 10 mg/kg/day, more specifically the 10 μg/kg/day to 1mg/kg/day, more specifically the 50 μg/kg/day to 100 μg/kg/day, but notlimited to these numbers and can vary according to age, health status,complications and other various factors.

The terms used herein is intended to be used to describe theembodiments, not to limit the present invention. Terms without numbersin front are not to limit the quantity but to show that there may bemore than one thing of the term used. The term “including”, “having”,“consisting”, and “comprising” shall be interpreted openly (i.e.“including but not limited to”).

Mention of range of numbers is used instead of stating separate numberswithin the range, so unless it is explicitly stated, each number can beread as separate numbers integrated herein. The end values of all rangesare included in the range and can be combined independently.

Unless otherwise noted or clearly contradicting in context, all methodsmentioned herein can be performed in the proper order. The use of anyone embodiment and all embodiment, or exemplary language (e.g., that use“like ˜”), unless included in the claims, is used to more clearlydescribe the present invention, not to limit the scope of the presentinvention. Any language herein outside of the claims should not beinterpreted as a necessity of the present invention. Unless definedotherwise, technical and scientific terms used herein have meaningnormally understood by a person skilled in the art that the presentinvention belongs to.

The preferred embodiments of the present invention are the best modeknown to the inventors to perform the present invention. It can becomeclear to those skilled in the art after reading the statements ahead ofthe variations in the preferred embodiments. The present inventors hopethat those skilled in the art can use the variations adequately andpresent invention be conducted in other ways than listed herein. Thus,the present invention, as allowed by the patent law, includesequivalents, and variations thereof, of the key points of the inventionstated in the appended claims. In addition, all possible variationswithin any combination of the above-mentioned components are included inthe present invention, unless explicitly stated otherwise orcontradicting in context. Although the present invention is describedand shown by exemplary embodiments, those skilled in the art willunderstand well that there can be various changes in the form anddetails without departing from the spirit of the invention and range,defined by the claims below.

Example 1: Synthesis of Peptide

The peptides of SEQ ID NO: 1 to SEQ ID NO: 156 were synthesizedaccording to the existing method of solid phase peptide synthesis. Indetail, the peptides were synthesized by coupling each amino acid fromC-terminus through Fmoc solid phase peptide synthesis, SPPS, usingASP48S (Peptron, Inc., Daejeon ROK). Those peptides with their firstamino acid at the C-terminus being attached to resin were used asfollows:

-   NH₂-Lys(Boc)-2-chloro-Trityl Resin-   NH₂-Ala-2-chloro-Trityl Resin-   NH₂-Arg(Pbf)-2-chloro-Trityl Resin

All the amino acid materials to synthesize the peptide were protected byFmoc at the N-terminus, and the amino acid residues were protected byTrt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl) that can be dissolved in acid. Such as:

-   Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH,    Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH,    Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH,    Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH,    Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

HBTU [2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminiumhexafluorophosphate]/HOBt [N-Hydroxybenzotriazole]/NMM[4-Methylmorpholine] were used as the coupling reagents. In 20% of DMF,piperidine was used to remove Fmoc. In order to remove the protectionfrom residue or to separate the synthesized peptide from Resin, cleavagecocktail [TFA (trifluoroacetic acid)/TIS (triisopropylsilane)/EDT(ethanedithiol)/H₂O=92.5/2.5/2.5/2.5] was used.

Peptides were synthesized by using the solid phase scaffold by addingeach amino acid with the sequential processes as follow; amino acidprotection, coupling reaction, washing, and deprotection. After cuttingoff the synthesized peptide from the resin, it was purified by HPLC andverify for synthesis by MS, and then freeze-dried.

Specific peptide synthesis process is described by the following withexample of Pep1 of SEQ ID NO: 164 (EARPALLTSRLRFIPK).

1) Coupling

The amino acid (8 equivalent) protected withNH₂-Lys(Boc)-2-chloro-Trityl Resin was melted in coupling agent HBTU (8equiv.)/HOBt (8 equiv.)/NMM (16 equiv.), and upon addition of DMF, thereaction mixture was incubated at room temperature for 2 hours, thenwashed sequentially with DMF, MeOH, and DMF.

2) Fmoc Deprotection

Following the addition of 20% piperidine in DMF, the reaction mixturewas incubated at room temperature for 5 minutes 2 times, then washedsequentially with DMF, MeOH, and DMF.

3) Make the Basic Framework of Peptide by Repeating Reactions 1 and 2Repeatedly.

4) Cleavage: Add Cleavage Cocktail to the Completely Synthesized Peptideand Separate the Peptide From the Resin.

5) Add pre-chilled diethyl ether into the mixture, and then centrifugethe reaction mixture to precipitate out the peptides.

6) After purification by Prep-HPLC, Check the Molecular Weight by LC/MSand Lyophilize to Obtain the Peptides in a Powder Form.

Example 2: Preparation of Pep(CPP)-FITC Conjugate

(1) Preparation of FITC-CPP Conjugate

A conjugate of the peptides with SEQ ID NO: 1 to SEQ ID NO: 156 combinedwith FITC was manufactured as follows, for example, a conjugate of pep1(SEQ ID NO: 157) and FITC, in other words, FITC-linker-pep1 wasmanufactured as follows.

The basic framework of peptide,NH₂-linker-E(OtBu)-A-R(Pbf)-P-A-L-L-T(tBu)-S(tBu)-R(PbOL-R(Pbf)-F-I-P-K(Boc)-2-chloro-TritylResin) which was obtained according to the manufacturing methodsdescribed in Example 1, was reacted with FITC. Specifically, FITC(fluorescein-5isothiocyanate) (8 equivalent) and DIPEA(N,N-Diisopropylethylamine) (16 equivalent) were melted in DMF. The DMFsolution was added, and reacted at room temperature for 2 hours, thenwashed sequentially with DMF, MeOH and DMF. As a result,FITC-linker-E(OtBu)-A-R(Pbf)-P-A-L-L-T(tBu)-S(tBu)-R(Pbf)L-R(Pbf)-F-I-P-K(Boc)-2-chloro-TritylResin was obtained. The linker herein is 6-aminohexanoic acid, Ahx.TFA/TIS/H₂O=95/2.5/2.5 was added to the peptide made on the resin, andthe conjugate was separated from the resin. A pre-chilled diethyl etherwas added to the obtained mixture, and centrifugation was used toprecipitate the peptide conjugates. After purification by Prep-HPLC,purity was confirmed with the analytical HPLC and the molecular weightwas determined by LC/MS. The peptide synthesized as described above wasverified as FITC-pep1 by confirmation of the molecular weight by LC/MS.Then the conjugates were lyophilized. SEQ ID NO: 1 to SEQ ID NO: 156 ofpeptides fused FITC with conjugate was also prepared as pep 1 of SEQ IDNO: 157.

(2) Preparation of a CPP-FITC Conjugate

The basic framework of the peptide,(NH₂-E(OtBu)-A-R(Pbf)-P-A-L-L-T(tBu)-S(tBu)-R(Pbf)L-R(Pbf)-F-I-P-K(Dde)-2-chloro-TritylResin) was generated according to the manufacturing methods described inthe Example 2 1. (1). To selectively introduce FITC to the C-term of thepeptide, the N-term of the peptide was protected from Boc. Then, theDi-tert-butyl dicarbonate (30 equivalent) and DIPEA (30 equivalent) weremelted in DMF. The DMF solution was added to the peptide and incubatedat room temperature for 2 hours, and the peptide was washed sequentiallywith DMF, MeOH, and DMF. As a result,Boc-E(OtBu)-A-R(Pbf)-P-A-L-L-T(tBu)-S(tBu)-R(PbOL-R(Pbf)-F-I-P-K(Dde)-2-chloro-TritylResin was obtained. Hydrazine in 2% of DMF was used to remove Dde whichis the protecting group of the C-terminal residue Lys in order to addFITC to the C-terminal of Lys. Then, FITC (8 equivalent) and DIPEA (16equivalent) were melted in DMF which was added to the peptide reactionmixture, and the mixture was incubated at room temperature for 2 hours,then washed sequentially with DMF, MeOH, DMF. As a result,Boc-E(OtBu)-A-R(Pbf)-P-A-L-L-T(tBu)-S(tBu)-R(Pbf)L-R(Pbf)-F-I-P-K(FITC)-2-chloro-TritylResin was obtained. TFA/TIS/H₂O=95/2.5/2.5 was added to separate thepeptide from resin. Pre-chilled diethyl ether was added to the mixture,and centrifugation was used to precipitate the peptides. Afterpurification by Prep-HPLC, purity was confirmed with the analytical HPLCand the molecular weight was confirmed with LC/MS. The obtainedsubstances were verified as pep1-FITC by confirmation of the molecularweight by LC/MS. Then the conjugates were lyophilized. SEQ ID NO: 1 toSEQ ID NO: 156 of peptide-FITC conjugates were also prepared in the samemanner as described above pep1-FITC.

Example 3: Experimental Cell Penetration of Pep(CPP)-FITC Conjugate

(1) Experimental Cell Penetration in HeLa Cell Line

Cell Culture

Homo sapiens cervix adenocarcinoma cell line as HeLa cell lines werepurchased from ATCC (American Type Cell Culture). The cells werecultured in MEM supplemented with 10% fetal bovine serum (Invitrogen,USA), Earle's salts, non-essential amino acids, sodium pyruvate and 100μg/ml penicillin and 10 units/ml streptomycin and cultured at 37° C., 5%CO₂ incubator.

Flow Cytometry and Confocal Microscopy Analysis of Cell Penetrating

Flow cytometry and Confocal microscope analysis were performed tocompare the between degree of cellular uptake of the cells were treatedwith SEQ ID NO: 1 to SEQ ID NO: 156, pep (CPP) and control.

The cell line was divided in a 6-well plate and cultured in a mediumcontaining 10% fetal bovine serum (Invitrogen, USA), 100 μg/mlpenicillin, 100 units/ml streptomycin at 37° C., 5% CO₂ incubator for 12hours. After washing the cell line with PBS, starvation was induced in aMinimum Essential Medium for an hour. 20 uM of each carrier peptide wastreated and cultured at 37° C. for an hour. After repeating the step ofwashing the cells with PBS for three times, Trypsin-EDTA was treatedform 10 mins at 37° C. to separate the carrier peptide on the outside ofthe cell. cells were collected with refrigerated PBS and centrifugationwas performed to repeat the step of washing the cells for three times.After then, the cells were suspended in 0.5 ml of PBS containing 4%Paraformaldehyde and fluorescence of the cells was analyzed using FACSCalibur (Becton Dickinson). The cellular uptake aspect of control andvarious peptides combined with FITC was compared and analyzed by MFI(Mean Fluorescence Intensity).

The results are as shown in FIGS. 1 to 23. Analysis results shown inFIG. 1 to FIG. 23 are given in detail in Table 3 below.

TABLE 3 pep2 pep3 pep4 pep5 pep6 PelP7 pep8 control 3.98 ± 0.87 2.39 ±0.23 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 peptide37.6 ± 1.89 18.23 ± 3.21  28.35 ± 2.54  19.06 ± 1.33  15.81 ± 1.59  9.51± 2.95 17.75 ± 1.96  pep11 pep12 pep13 pep14 pep15 pep6 pep16 control3.98 ± 0.87 2.39 ± 0.23 3.98 ± 0.87 3.98 ± 0.87 2.39 ± 0.23 3.98 ± 0.873.98 ± 0.87 peptide 559.78 ± 3.41  10.46 ± 1.99   22 ± 2.6 14.01 ± 1.32 10.51 ± 3.71  35.61 ± 1.29  20.12 ± 1.84  pep17 pep18 pep19 pep20 pep21pep22 pep23 control 3.98 ± 0.87 3.98 ± 0.87 2.39 ± 0.23 3.98 ± 0.87 2.39± 0.23 3.98 ± 0.87 3.98 ± 0.87 peptide 15.26 ± 2.23  18.7 ± 1.21 13.04 ±3.41  20.73 ± 2.45  13.22 ± 1.67  13.56 ± 0.23  15.68 ± 2.03  pep24pep25 pep26 pep27 pep28 pep29 pep30 control 3.98 ± 0.87 3.98 ± 0.87 3.98± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 peptide 13.78 ±1.07  18.62 ± 0.69  14.81 ± 1.77  9.89 ± 2.51 14.08 ± 3.11  33.5 ± 2.4814.37 ± 1.73  pep31 pep32 pep38 pep34 pep35 pep36 pep37 control 3.98 ±0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ±0.87 peptide 14.93 ± 2.05  11.98 ± 2.92  14.36 ± 1.63  14.41 ± 3.44 15.54 ± 1.38  15.37 ± 2.65  20.81 ± 1.02  pep38 pep39 pep40 pep41 pep42pep43 pep45 control 3.31 ± 0.62 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98± 0.87 3.98 ± 0.87 3.31 ± 0.62 peptide 380.4 ± 2.6  21.4 ± 0.99 11.63 ±1.11  28.97 ± 3.44  20.82 ± 1.93  29.88 ± 2.35  203.77 ± 3.26  pep46pep47 pep48 pep49 pep50 pep51 pep52 control 3.98 ± 0.87 3.98 ± 0.87 3.98± 0.87 3.98 ± 0.87 2.39 ± 0.23 3.98 ± 0.87 3.98 ± 0.87 peptide 24.67 ±2.02  15.85 ± 1.03  26.51 ± 2.36  13.91 ± 0.51  2228.76 ± 3.68   59.81 ±1.38  16.67 ± 2.22  pep53 pep54 pep56 pep57 pep58 pep59 pep60 control3.98 ± 0.87 2.52 ± 0.41 3.98 ± 0.87 2.39 ± 0.23 3.98 ± 0.87 3.98 ± 0.873.98 ± 0.87 peptide 16.16 ± 0.66  388.45 ± 2.94  28.61 ± 0.44  568.02 ±3.01  19.87 ± 0.94  18.58 ± 0.52  15.79 ± 1.63  pep62 pep63 pep65 pep66pep68 pep70 pep71 control 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 2.52 ±0.41 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 peptide 14.55 ± 3.71  21.3 ±1.55 12.72 ± 1.36  20.7 ± 2.57 27.28 ± 1.28  21.82 ± 1.76  16.61 ± 0.88 pep72 pep76 pep79 pep80 pep81 pep82 pep83 control 3.98 ± 0.87 2.52 ±0.41 3.98 ± 0.87 3.98 ± 0.87 2.39 ± 0.23 3.98 ± 0.87 3.98 ± 0.87 peptide17.57 ± 2.31  13.98 ± 0.11  31.2 ± 1.52 12.45 ± 0.46  29.69 ± 2.49 22.44 ± 3.32  24.57 ± 1.36  pep87 pep88 pep89 pep91 pep92 pep94 pep95control 2.52 ± 0.41 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 2.39± 0.23 3.98 ± 0.87 peptide 38.35 ± 2.27  16.43 ± 0.55  13.54 ± 0.75 24.41 ± 2.79  19.91 ± 0.51  117.76 ± 4.24  10.92 ± 0.07  pep96 pep102pep103 pep104 pep105 pep106 pep107 control 2.39 ± 0.23 2.52 ± 0.41 3.98± 0.87 2.39 ± 0.23 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 peptide 308.53 ±3.61  45.84 ± 1.58  126.88 ± 2.14  36.74 ± 1.11  13.23 ± 2.21  12.45 ±0.38  23.11 ± 0.56  pep108 pep109 pep110 pep113 pep116 pep117 pep118control 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98 ± 0.87 3.98± 0.87 2.53 ± 0.17 peptide 22.53 ± 0.95  12.46 ± 1.32  22.04 ± 0.77 16.09 ± 2.5  20.5 ± 0.97 23.11 ± 2.46  14.69 ± 0.33  pep119 pep123pep124 pep125 pep126 pep128 pep131 control 2.53 ± 0.17 3.98 ± 0.87 2.53± 0.17 2.53 ± 0.17 2.53 ± 0.17 3.98 ± 0.87 2.39 ± 0.23 peptide 13.23 ±0.64  18.03 ± 2.83  23.74 ± 3.62  28.62 ± 2.12  3362.29 ± 3.24   14.36 ±1.21  32.51 ± 2.38  pep132 pep133 pep134 pep138 pep139 pep141 pep145control 3.98 ± 0.87 2.53 ± 0.17 2.39 ± 0.23 3.98 ± 0.87 2.39 ± 0.23 3.98± 0.87 3.98 ± 0.87 peptide 26.39 ± 0.95  46.79 ± 1.92  78.05 ± 2.26 16.43 ± 0.91  29.95 ± 0.28  20.89 ± 0.31  11.97 ± 0.78  pep146 pep150pep151 pep153 pep158 pep160 control 2.53 ± 0.17 3.98 ± 0.87 3.98 ± 0.872.53 ± 0.17 3.98 ± 0.87 2.53 ± 0.17 peptide 18.32 ± 1.82  17.46 ± 0.24 23.52 ± 0.56  31.72 ± 1.74  87.75 ± 2.51  9.860.23

(2) Cell Penetrating Property in Huh7 Cell Line

Cell Culture

human hepatocellular carcinoma cell line as Huh7 lines were purchasedfrom ATCC

(American Type Cell Culture) and used as suspended cells. The cells werecultured in MEM supplemented with 10% fetal bovine serum (Invitrogen,USA), EarleSA), Earle Huh7 lines were purchased from ATpyruvate and 100μa/ml penicillin and 10 units/ml streptomycin and cultured at 37° C., 5%CO₂ incubator.

The Screening Analysis of Cell Penetrating Used by Flow Cytometry

To confirm the cell penetrating of peptides, the Huh7 cell lines weretreated with SEQ ID NO: 1 to SEQ ID NO: 156 and analysed by Flowcytometry. The analysed method was also confirmed by the same manner asdescribed above example (1) in Hela cell lines. Also, the result showedin FIG. 24 to FIG. 43.

(3) Experimental Cell Penetration in Human T Lymphocyte Cell Lines

Cell Culture

human T-cell leukemia cell line as Jurket was purchased from ATCC(American Type Cell Culture) and used as suspended cells. The cells werecultured in RPMI 1640 supplemented with 10% fetal bovine serum(Invitrogen, USA), EarleSA), EarleC (American Type Cell Culture) andpyruvate and 100 om ATCC (American Type units/ml streptomycin andcultured at 37° C., 5% CO₂ incubator. Human derived lymphocytes(lymphocyte) separated from the healthy human blood (50 m) and thencollected layer of peripheral blood mononuclear cells (PBMC) andlymphocytes used by Biocoll separating solution (Biochrom AG, Berlin,Germany).

The Screening Analysis of Cell Penetrating Used by Flow Cytometry

To confirm the cell penetrating of peptides, the human T-cell leukemiacell lines were treated with SEQ ID NO: 1 to SEQ ID NO: 156 and analysedby Flow cytometry. The analysed method was also confirmed by the samemanner as described above example (1) in Hela cell lines. Also, theresult showed in FIG. 44 to FIG. 58.

(4) Analysis of Cell Viability and Toxicity

The cell line was divided into 96-well plate and cultured in a mediumcontaining 10% fetal bovine serum (Invitrogen, USA), 100 μg/mlpenicillin, 100 units/ml streptomycin at 37° C., 5% CO₂ incubator for 12hours. After washing the cell line with PBS, starvation was induced in aMinimum Essential Medium for an hour. 20 uM of each carrier peptide wastreated and cultured at 37° C. for an hour. After cultured cells,analysed cell viability and toxicity used by MTT assay. The resultsshowed in FIG. 59 to FIG. 72.

What is claimed is:
 1. A conjugate consisting of a mitochondrialtargeting peptide and ferrocene carboxylate, wherein said mitochondrialtargeting peptide consists of 30 amino acids or less.
 2. The conjugateof claim 1, wherein the mitochondrial targeting peptide is derived fromhuman telomerase reverse transcriptase (hTERT) enzyme.
 3. The conjugateof claim 2, wherein the mitochondrial targeting peptide comprises SEQ IDNO:
 48. 4. The conjugate of claim 1, wherein the mitochondrial targetingpeptide and ferrocene carboxylate are combined via a covalent bond. 5.The conjugate of claim 1, wherein the mitochondrial targeting peptideand ferrocene carboxylate are combined via a non-covalent bond.
 6. Acontrasting agent comprising the conjugate of claim
 1. 7. A compositioncomprising the conjugate of claim 1 and a pharmaceutically acceptablecarrier.